Β-galactosidase Activity Assay Research Articles

Isolation and characterization of active promoters from Gluconacetobacter diazotrophicus strain PAL5 using a promoter-trapping plasmid.

Gluconacetobacter diazotrophicus is a nitrogen-fixing, endophytic bacterium that has the potential to promote plant growth and increase yield. Genetically modified strains might get more benefits to host plants, including through expression of useful proteins, such as Cry toxins from B. thuringiensis, or enzymes involved in phytohormone production, proteins with antagonistic activity for phytopathogens, or that improve nutrient utilization by the plant. For that, expression systems for G. diazotrophicus are needed, which requires active promoters fused to foreign (or innate) genes. This article describes the construction of a G. diazotrophicus PAL5 promoter library using a promoter-less lacZ-bearing vector, and the identification of six active promoters through β-galactosidase activity assays, sequencing and localization in the bacterial genome. The characterized promoters, which are located on distinct regions of the bacterial genome and encoding either sense or antisense transcripts, present variable expression strengths and might be used in the future for expressing useful proteins.

Inhibitory effect of UvrD and DinG on the replication of ColE1-derived plasmids in Escherichia coli

CspA has been identified as a major cold-shock protein in Escherichia coli. CspA binds to RNAs which are abnormally folded at low temperature and then acts as an RNA chaperone unfolding those RNAs. The dramatic expression of cspA at low temperature is contributed by posttranscriptional stability and robust translatability. Interestingly, when cspA mRNA encoding a premature nonsense codon was overexpressed at low temperature, cell growth was completely inhibited. This phenotype was termed LACE (the low temperature-dependent antibiotic effect of truncated cspA expression), and this lethality resulted from exclusive stalling of most ribosomes on mutant cspA mRNAs. In a previous study, we demonstrated that overexpression of the ATP-dependent DNA helicases, UvrD and DinG, suppressed the lethality and ribosome stalling caused by mutant cspA mRNA. In the present study, we attempted to elucidate how these two DNA helicases help recover normal growth under LACE condition. Interestingly, we found that UvrD and DinG appeared to have an ability to down-regulate the replication of pUC-based high copy plasmid. In plasmid copy number tests, the amount of pUC-based plasmid encoding mutant cspA was reduced by 3–10-fold when either UvrD or DinG was expressed. Through a β-galactosidase activity assay, we also confirmed that expression of the lacZα gene inserted into the pUC-based plasmid was significantly reduced due to down-regulation of plasmid replication. Our findings imply that UvrD and DinG, known as non-replicative helicases, play a novel role in the regulation of ColE1-like plasmid replication.

Genome-wide analysis of AP2/ERF transcription factors in carrot (Daucus carota L.) reveals evolution and expression profiles under abiotic stress.

AP2/ERF is a large transcription factor family that regulates plant physiological processes, such as plant development and stress response. Carrot (Daucus carota L.) is an important economical crop with a genome size of 480Mb; the draft genome sequencing of this crop has been completed by our group. However, little is known about the AP2/ERF factors in carrot. In this study, a total of 267 putative AP2/ERF factors were identified from the whole-genome sequence of carrot. These AP2/ERF proteins were phylogenetically clustered into five subfamilies based on their similarity to the amino acid sequences from Arabidopsis. The distribution and comparative genome analysis of the AP2/ERF factors among plants showed the AP2/ERF factors had expansion during the evolutionary process, and the AP2 domain was highly conserved during evolution. The number of AP2/ERF factors in land plants expanded during their evolution. A total of 60 orthologous and 145 coorthologous AP2/ERF gene pairs between carrot and Arabidopsis were identified, and the interaction network of orthologous genes was constructed. The expression patterns of eight AP2/ERF family genes from each subfamily (DREB, ERF, AP2, and RAV) were related to abiotic stresses. Yeast one-hybrid and β-galactosidase activity assays confirmed the DRE and GCC box-binding activities of DREB subfamily genes. This study is the first to identify and characterize the AP2/ERF transcription factors in carrot using whole-genome analysis, and the findings may serve as references for future functional research on the transcription factors in carrot.

A novel regulator PA5022 (aefA) is involved in swimming motility, biofilm formation and elastase activity of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen contributing to a range of nosocomial infections. To identify new genes involved in P. aeruginosa swimming motility, an important mechanism of pathogenesis, mutants with altered swimming motility patterns from Mu transposon mutagenesis library of the P. aeruginosa clinical strain PA68 were isolated and characterized. We identified a mutant with transposon inactivation of PA5022 has completely abolished its swimming motility while still possesses a normal terminal flagellum according to electronic microscopy analysis. Microscopic examination revealed that the PA5022 mutant forms thicker biofilms compared to the PA68 wild-type strain and is impaired in its elastase activity. To exclude the possibility of genetic diversity in affecting gene functions among different strains, we constructed a PA5022 knock out mutant based on the PAK lab strain. The PAKΔPA5022 has similar phenotypes to the PA5022 (PA5022::Mu) mutant of PA68 strain. Furthermore, transcriptional fusion assays were carried out to investigate the regulatory mechanism of PA5022 by using the PlasI-lacZ, PrhlI-lacZ, PrpoN-lacZ, PrpoS-lacZ, PqscR-lacZ, PvqsR-lacZ fusions. β-Galactosidase activity assays indicated that the expression of the vqsR, lasI and rhlI promotors was reduced in the PA5022 mutant compared to the PA68 wild-type. Our study showed that PA5022 links swimming motility and quorum sensing, which might be an important regulator for the pathogenesis of P. aeruginosa.

Connective tissue growth factor differentially binds to members of the cystine knot superfamily and potentiates platelet-derived growth factor-B signaling in rabbit corneal fibroblast cells.

To study the binding of connective tissue growth factor (CTGF) to cystine knot-containing ligands and how this impacts platelet-derived growth factor (PDGF)-B signaling. The binding strengths of CTGF to cystine knot-containing growth factors including vascular endothelial growth factor (VEGF)-A, PDGF-B, bone morphogenetic protein (BMP)-4, and transforming growth factor (TGF)-β1 were compared using the LexA-based yeast two-hybrid system. EYG48 reporter strain that carried a wild-type LEU2 gene under the control of LexA operators and a lacZ reporter plasmid (p80p-lacZ) containing eight high affinity LexA binding sites were used in the yeast two-hybrid analysis. Interactions between CTGF and the tested growth factors were evaluated based on growth of transformed yeast cells on selective media and colorimetric detection in a liquid β-galactosidase activity assay. Dissociation constants of CTGF to VEGF-A isoform 165 or PDGF-BB homo-dimer were measured in surface plasma resonance (SPR) analysis. CTGF regulation in PDGF-B presentation to the PDGF receptor β (PDGFRβ) was also quantitatively assessed by the SPR analysis. Combinational effects of CTGF protein and PDGF-BB on activation of PDGFRβ and downstream signaling molecules ERK1/2 and AKT were assessed in rabbit corneal fibroblast cells by Western analysis. In the LexA-based yeast two-hybrid system, cystine knot motifs of tested growth factors were fused to the activation domain of the transcriptional factor GAL4 while CTGF was fused to the DNA binding domain of the bacterial repressor protein LexA. Yeast co-transformants containing corresponding fusion proteins for CTGF and all four tested cystine knot motifs survived on selective medium containing galactose and raffinose but lacking histidine, tryptophan, and uracil. In liquid β-galactosidase assays, CTGF expressing cells that were co-transformed with the cystine knot of VEGF-A had the highest activity, at 29.88 ± 0.91 fold above controls (P < 0.01). Cells containing the cystine knot of BMP-4 expressed the second most activity, with a 24.77 ± 0.47 fold increase (P < 0.01). Cells that contained the cystine knot of TGF-β1 had a 3.80 ± 0.66 fold increase (P < 0.05) and the ones with the cystine knot of PDGF-B had a 2.64 ± 0.33 fold increase of β-galactosidase activity (P < 0.01). Further SPR analysis showed that the association rate between VEGF-A 165 and CTGF was faster than PDGF-BB and CTGF. The calculated dissociation constant (KD) of CTGF to VEGF165 and PDGF-BB was 1.8 and 43 nmol/L respectively. PDGF-BB ligand and PDGFRβ receptor formed a stable complex with a low dissociation constant 1.4 nmol/L. Increasing the concentration of CTGF up to 263.2 nmol/L significantly the ligand/receptor binding. In addition, CTGF potentiated phosphorylation of PDGFRβ and AKT in rabbit corneal fibroblast cells stimulated by PDGF-BB in tissue culture condition. In contrast, CTGF did not affect PDGF-B induced phosphorylation of ERK1/2. CTGF has a differential binding affinity to VEGF-A, PDGF-B, BMP-4, and TGF-β. Its weak association with PDGF-B may represent a novel mechanism to enhance PDGF-B signaling.

A TetR Family Transcriptional Factor Directly Regulates the Expression of a 3-Methyladenine DNA Glycosylase and Physically Interacts with the Enzyme to Stimulate Its Base Excision Activity in Mycobacterium bovis BCG

3-Methyladenine DNA glycosylase recognizes and excises a wide range of damaged bases and thus plays a critical role in base excision repair. However, knowledge on the regulation of DNA glycosylase in prokaryotes and eukaryotes is limited. In this study, we successfully characterized a TetR family transcriptional factor from Mycobacterium bovis bacillus Calmette-Guerin (BCG), namely BCG0878c, which directly regulates the expression of 3-methyladenine DNA glycosylase (designated as MbAAG) and influences the base excision activity of this glycosylase at the post-translational level. Using electrophoretic mobility shift assay and DNase I footprinting experiments, we identified two conserved motifs within the upstream region of mbaag specifically recognized by BCG0878c. Significant down-regulation of mbaag was observed in BCG0878c-overexpressed M. bovis BCG strains. By contrast, about 12-fold up-regulation of mbaag expression was found in bcg0878c-deleted mutant M. bovis BCG strains. β-Galactosidase activity assays also confirmed these results. Thus, BCG0878c can function as a negative regulator of mbaag expression. In addition, the regulator was shown to physically interact with MbAAG to enhance the ability of the glycosylase to bind damaged DNA. Interaction between the two proteins was further found to facilitate AAG-catalyzed removal of hypoxanthine from DNA. These results indicate that a TetR family protein can dually regulate the function of 3-methyladenine DNA glycosylase in M. bovis BCG both at the transcriptional and post-translational levels. These findings enhance our understanding of the expression and regulation of AAG in mycobacteria.

Screening and identification of ClpE interaction proteins in Streptococcus pneumoniae by a bacterial two-hybrid system and co-immunoprecipitation

Hsp100/Clp proteins have crucial functions in the protein quality control, stress tolerance, and virulence of many pathogenic bacteria. ClpE is an important virulence factor involved in adherence and invasion in Streptococcus pneumoniae. To explore the underlying mechanism, we screened ClpE interaction proteins using a bacterial two-hybrid system and co-immunoprecipitation. We used ClpE as bait and constructed the pBT-ClpE bait plasmid for two-hybrid screening. Then, we constructed ClpE::GFP fusion for co-immunoprecipitation screening using anti-GFP monoclonal antibody. We obtained eight potential ClpE interaction proteins, including carbamoyl-phosphate synthase, pyruvate oxidase (SpxB), phosphoenolpyruvate-protein phosphotransferase, aminopeptidase N (pepN), L-lactate dehydrogenase, ribosomal protein S4, sensor histidine kinase (SPD_2019), and FtsW (a cell division protein). FtsW, SpxB, pepN, and SPD_2019 were confirmed to interact with ClpE using Bacterial Two-hybrid or Co-immunoprecipitation. Morphologic observations found that ΔclpE strain existed in abnormal division. β-Galactosidase activity assay suggested that ClpE contributed to the degradation of FtsW. Furthermore, FtsW could be induced by heat shock. The results suggested that ClpE might affect cell division by regulating the level of FtsW. These data may provide new insights in studying the role of ClpE in S. pneumoniae.

754 Tumorigenesis Promoted by Colibactin-Producing Escherichia coli Explained by the Dark Side of Cellular Senescence

Background & Aims: Microbiota has been shown to play a critical role in colorectal cancer (CRC). Most of CRC mucosa-associated Escherichia coli harbor the pks genomic island (pks+ E. coli) responsible for the synthesis of colibactin, a genotoxic compound able to promote colon tumorigenesis in a genetically susceptible mouse model. Our aim was to investigate the mechanisms underlying the tumorigenic activity of E. coli. Methods: Human intestinal epithelial cells HCT116 were infected with pks+ or pksE. coli. Cellular senescence and proliferation were assessed by β-galactosidase activity and MTT assay, respectively. Effect of growth factors secreted from infected cells on the proliferation of uninfected cells was assessed using specific neutralizing antibodies and inhibitors in vitro and in a xenograft mouse model. Human biopsies isolated from CRC tumors colonized with pks+ or pksE. coli were used to validate the experimental data. Results: A transient infection of HCT16 cells with pks+ E. coli increased tumor growth in the xenograft mouse model compared to pksE. coli infection, and the conditioned media derived from pks+ E. coli-infected cells stimulated the growth of uninfected cells. A pretreatment of the conditioned media with an anti-HGF antibody and inhibition of HGF receptor in the xenograft mouse model abolished the pro-proliferative effect of pks+ E. coli, indicating the involvement of the growth factor HGF. HGF was produced by pks+ E. coli-induced senescent cells, which is driven by posttranslational modification of p53 involving small ubiquitin-like modifier (SUMO) peptides. The pks+ E. coli-infected cells displayed a modified pattern of SUMO-conjugated proteins and decreased expression of the SUMO peptidase SENP1, a key regulator of SUMOylation. Over-expression of SENP1 in HCT116 cells abolished the deregulated SUMOylation, cellular senescence, HGF production and the pro-proliferative effect induced by pks+ E. coli infection. Finally, the human CRC tumors colonized by pks+ E. coli expressed a higher level of HGF and decreased SENP1 expression compared to those colonized by pksE. coli, supporting the pathological relevance of the in vitro data. Conclusions: Our study elucidates a new paradigm for the mechanism underlying the carcinogenesis mediated by colibactin-producing bacteria. We propose that those bacteria modulate tumor microenvironment by favoring the emergence of senescent cells, which in turn promote tumor growth by the secretion of growth factors, notably HGF.

757 Impact of Metabolic Syndrome on Incident or Recurrent Advanced Adenoma After Colonoscopy

Background & Aims: Microbiota has been shown to play a critical role in colorectal cancer (CRC). Most of CRC mucosa-associated Escherichia coli harbor the pks genomic island (pks+ E. coli) responsible for the synthesis of colibactin, a genotoxic compound able to promote colon tumorigenesis in a genetically susceptible mouse model. Our aim was to investigate the mechanisms underlying the tumorigenic activity of E. coli. Methods: Human intestinal epithelial cells HCT116 were infected with pks+ or pksE. coli. Cellular senescence and proliferation were assessed by β-galactosidase activity and MTT assay, respectively. Effect of growth factors secreted from infected cells on the proliferation of uninfected cells was assessed using specific neutralizing antibodies and inhibitors in vitro and in a xenograft mouse model. Human biopsies isolated from CRC tumors colonized with pks+ or pksE. coli were used to validate the experimental data. Results: A transient infection of HCT16 cells with pks+ E. coli increased tumor growth in the xenograft mouse model compared to pksE. coli infection, and the conditioned media derived from pks+ E. coli-infected cells stimulated the growth of uninfected cells. A pretreatment of the conditioned media with an anti-HGF antibody and inhibition of HGF receptor in the xenograft mouse model abolished the pro-proliferative effect of pks+ E. coli, indicating the involvement of the growth factor HGF. HGF was produced by pks+ E. coli-induced senescent cells, which is driven by posttranslational modification of p53 involving small ubiquitin-like modifier (SUMO) peptides. The pks+ E. coli-infected cells displayed a modified pattern of SUMO-conjugated proteins and decreased expression of the SUMO peptidase SENP1, a key regulator of SUMOylation. Over-expression of SENP1 in HCT116 cells abolished the deregulated SUMOylation, cellular senescence, HGF production and the pro-proliferative effect induced by pks+ E. coli infection. Finally, the human CRC tumors colonized by pks+ E. coli expressed a higher level of HGF and decreased SENP1 expression compared to those colonized by pksE. coli, supporting the pathological relevance of the in vitro data. Conclusions: Our study elucidates a new paradigm for the mechanism underlying the carcinogenesis mediated by colibactin-producing bacteria. We propose that those bacteria modulate tumor microenvironment by favoring the emergence of senescent cells, which in turn promote tumor growth by the secretion of growth factors, notably HGF.

755 Obesity Accelerates Helicobacter felis-Induced Gastric Carcinogenesis by Enhancing TH17 Responses and Immature Myeloid Cell Trafficking

Background & Aims: Microbiota has been shown to play a critical role in colorectal cancer (CRC). Most of CRC mucosa-associated Escherichia coli harbor the pks genomic island (pks+ E. coli) responsible for the synthesis of colibactin, a genotoxic compound able to promote colon tumorigenesis in a genetically susceptible mouse model. Our aim was to investigate the mechanisms underlying the tumorigenic activity of E. coli. Methods: Human intestinal epithelial cells HCT116 were infected with pks+ or pksE. coli. Cellular senescence and proliferation were assessed by β-galactosidase activity and MTT assay, respectively. Effect of growth factors secreted from infected cells on the proliferation of uninfected cells was assessed using specific neutralizing antibodies and inhibitors in vitro and in a xenograft mouse model. Human biopsies isolated from CRC tumors colonized with pks+ or pksE. coli were used to validate the experimental data. Results: A transient infection of HCT16 cells with pks+ E. coli increased tumor growth in the xenograft mouse model compared to pksE. coli infection, and the conditioned media derived from pks+ E. coli-infected cells stimulated the growth of uninfected cells. A pretreatment of the conditioned media with an anti-HGF antibody and inhibition of HGF receptor in the xenograft mouse model abolished the pro-proliferative effect of pks+ E. coli, indicating the involvement of the growth factor HGF. HGF was produced by pks+ E. coli-induced senescent cells, which is driven by posttranslational modification of p53 involving small ubiquitin-like modifier (SUMO) peptides. The pks+ E. coli-infected cells displayed a modified pattern of SUMO-conjugated proteins and decreased expression of the SUMO peptidase SENP1, a key regulator of SUMOylation. Over-expression of SENP1 in HCT116 cells abolished the deregulated SUMOylation, cellular senescence, HGF production and the pro-proliferative effect induced by pks+ E. coli infection. Finally, the human CRC tumors colonized by pks+ E. coli expressed a higher level of HGF and decreased SENP1 expression compared to those colonized by pksE. coli, supporting the pathological relevance of the in vitro data. Conclusions: Our study elucidates a new paradigm for the mechanism underlying the carcinogenesis mediated by colibactin-producing bacteria. We propose that those bacteria modulate tumor microenvironment by favoring the emergence of senescent cells, which in turn promote tumor growth by the secretion of growth factors, notably HGF.

Abstract 555: Proliferation, apoptosis and senescence differential modulation by combining the retinoid ATRA and Protein Kinase C pharmacological inhibitors, in a bicellular murine mammary tumor cell line.

Abstract Retinoids exert some of their effects on cell differentiation and malignant phenotype reversion through interactions with specific Protein Kinase C (PKC) isoforms. In this work we studied: A)- the modulation of PKCα and PKCδ expression when cells were treated with ATRA (1 μM), B)- The expression profile of Retinoic Acid Receptors (RAR) when cells are treated with ATRA and/or PKCα / PKC δ inhibitors (Gö6976 2.5 μM / Rottlerin 1 μM) and C)- the effect of combining ATRA and PKC inhibitors on in vitro cell growth, cell cycle distribution and senescence. We used as model the murine mammary tumor-derived cell line LM38-LP, composed by luminal (LEP) and myoepithelial (MEP) cells. By RT-PCR we could determine that 48 h ATRA treatment increased PKCδ only in the LM38-LP LEP population and decreased PKCα expression levels in both cellular components. Regarding RARs profile, the treatment with PKCα inhibitor Gö6976 reduced RARγ2 levels, while its combination with ATRA also increased RARβ2. Furthermore, 96 h treatment with ATRA, Gö6976, or Rottlerin or their combinations induced cell growth inhibition (cell number x105: control 22± 1.1; ATRA 11±0.1; Gö6976 3.6±0.6; Rottlerin 9±0.1; ATRA/Gö6976 2.3±0.03; ATRA/Rottlerin 6.7±1.3, p ≤0.05 vs control in all treatments). By flow cytometry we could determine that both ATRA and Rottlerin treatments induce LM38-LP cell cycle arrest in G1 phase, while Gö6976 increased the number of cells in the subG1 phase, correlating with increased apoptosis and confirmed by Annexin V-FITC staining. Employing β-galactosidase activity assay and bright field microscopy, we determined that 96 h ATRA treatment induced senescence mainly in the MEP component while Gö6976 does it in the LEP population. Interestingly, Rottlerin treated cells showed morphological alterations compatible with autophagy. We conclude that cell growth inhibition exerted by retinoids may be enhanced by the reduction in PKCα expression and / or activity. In addition the combination of ATRA plus Gö6976, induced differential responses on the different LM38-LP cell components, showing senescence in MEP cells, together with cell cycle arrest and apoptosis mainly in the LEP compartment. Consistently, all these biological effects correlated with an increase in RARβ2 (known to induce cell differentiation) and a decrease in RARγ2 (implicated in proliferation) mRNAs level. Citation Format: Damian E. Berardi, Maria I. Diaz Bessone, Carolina Flumian, Stefano M. Cirigliano, Elisa D. Bal de Kier Joffe, Alejandro J. Urtreger, Laura B. Todaro. Proliferation, apoptosis and senescence differential modulation by combining the retinoid ATRA and Protein Kinase C pharmacological inhibitors, in a bicellular murine mammary tumor cell line. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 555. doi:10.1158/1538-7445.AM2013-555

Day-long reduction of oral malodor by a palatal mucoadhesive tablet containing herbal formulation

Previous research has shown the oral malodor reducing abilities of an herbal formulation delivered using a palatal mucoadhesive tablet. The aim of the present study was to test the day-long effect of this preventive treatment on oral malodor reduction as compared with placebo and commercial mouthwash. Forty young healthy subjects (mean age, 25.8 ± 1.8 yrs, 19 females) presenting with oral malodor were randomly assigned to use one of the three tested products: herbal mucoadhesive tablets, placebo mucoadhesive tablets and a commercial mouthwash. Following baseline measurements, subjects were instructed to use the products in the evening of the same day and the following morning. Baseline and follow-up measurements were conducted in the afternoon and included odor judge scores (two judges), volatile sulfide compounds (VSC) levels using a sulfide monitor (Halimeter™) and saliva sample for β-galactosidase activity assay. The herbal mucoadhesive tablet caused a significant reduction in malodor scores (p = 0.004), VSC levels (p = 0.002) and β-galactosidase assay (p = 0.02) as compared to the placebo, and reduced malodor level to below the clinical threshold (mean odor judge score of 1.7). These results demonstrate the efficacy of the herbal formulation delivered using a mucoadhesive tablet for day-long prevention of oral malodor.

Altered Fate of Tendon-Derived Stem Cells Isolated from a Failed Tendon-Healing Animal Model of Tendinopathy

We hypothesized that altered fate of tendon-derived stem cells (TDSCs) might contribute to chondro-ossification and failed healing in the collagenase-induced (CI) tendon injury model. This study aimed to compare the yield, proliferative capacity, immunophenotypes, senescence, and differentiation potential of TDSCs isolated from healthy (HT) and CI tendons. TDSCs were isolated from CI and healthy Sprague-Dawley rat patellar tendons. The yield, proliferative capacity, immunophenotypes, and senescence of TDSCs (CI) and TDSCs (HT) were compared by colony-forming unit assay, BrdU assay, flow cytometry, and β-galactosidase activity assay, respectively. Their osteogenic and chondrogenic differentiation potentials and mRNA expression of tendon-related markers were compared using standard assays. More TDSCs, which showed a lower proliferative potential and a higher cellular senescence were present in the CI patellar tendons compared to HT tendons. There was a higher alkaline phosphatase activity and mineralization in TDSCs (CI) in both basal and osteogenic media. More chondrocyte-like cells and higher proteoglycan deposition, Sox9 and collagen type II expression were observed in TDSCs (CI) pellets upon chondrogenic induction. There was a higher protein expression of Sox9, but a lower mRNA expression of Col1a1, Scx, and Tnmd in TDSCs (CI) in a basal medium. In conclusion, TDSCs (CI) showed altered fate, a higher cellular senescence, but a lower proliferative capacity compared to TDSCs (HT), which might contribute to pathological chondro-ossification and failed tendon healing in this animal model.

Scanning assay of β-galactosidase activity

Beta-galactosidase, encoded by the lacZ gene in E. coli, can cleave lactose and structurally related compounds to galactose and glucose or structurally related products. Its activity can be measured using an artificial substrate, o-nitrophenyl-beta-D-galactopyranoside (ONPG). Miller firstly described the standard quantitative assay of beta-galactosidase activity in the cells of bacterial cultures by disrupting the cell membrane with the permeabilization solution instead of preparing cell extracts. Therefore, beta-galactosidase became one of the most widely used reporters of gene expression in molecular biology to reflect intracellular gene expression difference. But the Miller assay procedure could not monitor the beta-galactosidase reaction in real time and its results were greatly influenced by some operations in the Miller procedure, such as permeabilization time, reaction time and concentration of the cell suspension. A scanning method based on the Miller method to determine the intracellular beta-galactosidase activity in E. coli Tuner (DE3) expressing -galactosidase in real time was developed and the permeabilization time of cells was optimized for that. The comparison of 3 assays of beta-galactosidase activity (Miller, colorimetric and scanning) was made. The results proved that scanning method for the determination of enzyme activity with using ONPG as substrate is simple, fast and reproducible.

Accumulation of annexin A5 at the nuclear envelope is a biomarker of cellular aging

Cellular senescence is a permanent cell cycle arrest induced by short telomeres or oncogenic stress in vitro and in vivo. Because no single of the established biomarkers can reliably identify senescent cells, the application of new ones may aid the diagnosis of aged cells. Here we show that annexin A5 accumulates at the nuclear envelope during replicative and drug-induced cellular senescence in primary human fibroblasts. This new cellular aging phenotype that we have termed SA-ANX5 (senescence-associated accumulation at the nuclear envelope of annexin A5) is as efficient and quantitative as the well-established senescence-associated β-galactosidase activity assay and p21 immunoreactivity. SA-ANX5 is also observed in aged human skin where is exclusively detected in DNA damage foci-positive/Ki-67-negative cells. We also observed that depletion of annexin A5 by siRNA in human fibroblasts accelerates premature senescence through the p38MAP kinase pathway. These observations establish SA-ANX5 as a new biomarker for cellular aging and implicate a functional role for annexin A5 in cellular senescence.

Senescence of fetal endothelial progenitor cell in pregnancy with idiopathic fetal growth restriction

Objective: The aim of our study was to investigate the change of count and the status of cellular senescence in fetal endothelial progenitor cells (EPCs) obtained from the umbilical cord blood of women with fetal growth restriction (FGR). Methods: Fetal EPCs were obtained from thirty five normal and thirty pregnant women with FGR. Each EPC was characterized and counted. EPC differentiation time and outgrowth endothelial cell (OEC) colony formation assay, senescence-associated β-galactosidase (SA-β-gal) activity assay, and telomerase activity assay were performed. Results: Fetal EPC counts were significantly decreased in the FGR group compared with normal controls. In the FGR group, the EPC differentiation time was prolonged, OEC colonies were much less formed, the staining intensity of SA-β-gal was relatively increased and the telomerase activity of EPCs was significantly decreased, compared with normal pregnancy (p < 0.001 for all). Conclusions: The fetal EPCs in FGR pregnancies were decreased, functionally impaired and senescently altered.

Generation of Sperms Containing EGFP-LacZ Following Transfection of Chicken Testis with a Eukaryotic Dual Reporter Vector

Male germ cells modified by foreign genes can be used to generate transgenic chicken. In this study, in vivo transfection of chicken testis with an EGFP-LacZ dual reporter expression vector was performed. Large-scale plasmid DNA preparation of the EGFP-LacZ eukaryotic expression vector was carried out and efficient transfection of chicken testicle cells using the prepared plasmid DNA was confirmed in vitro. The reporter plasmid was directly injected into adult rooster testes. Semen samples were collected on 10-days post-transfection and every other day thereafter; and a total of six collections were made. Semen slides were subjected to fluorescence microscopy and β-galactosidase activity assay to identify sperms carrying the reporter genes. The presence of EGFP and LacZ was further confirmed by PCR amplification with sperm genomic DNA as template. The testicles of those birds were subjected to cryostat sectioning, fluorescence microscopy and β-galactosidase activity assay. The results showed that sperms with green fluorescence were not observed on semen slides; however, sperms positive for β-galactosidase were detected. Specific amplicons of EGFP and lacZ were detected in four of the six sequentially collected semen samples. Fluorescence microscopy of the corresponding semen slides revealed yellow-green fluorescence, but not clear green fluorescence. The β-galactosidase activity assay and GFP histochemistry using monoclonal antibodies demonstrated positive staining for subsets of testicle cells. Together, these results showed that direct injection of the dual reporter vector into adult rooster testis allowed in vivo transfection of chicken sperm precursor cells, which further developed into sperm containing EGFP-LacZ.

YEAST two-hybrid and itc studies of alpha and beta spectrin interaction at the tetramerization site

Yeast two-hybrid (Y2H) and isothermal titration calorimetry (ITC) methods were used to further study the mutational effect of non-erythroid alpha spectrin (αII) at position 22 in tetramer formation with beta spectrin (βII). Four mutants, αII-V22D, V22F, V22M and V22W, were studied. For the Y2H system, we used plasmids pGBKT7, consisting of the cDNA of the first 359 residues at the N-terminal region of αII, and pGADT7, consisting of the cDNA of residues 1697–2145 at the C-terminal region of βII. Strain AH109 yeast cells were used for colony growth assays and strain Y187 was used for β-galactosidase activity assays. Y2H results showed that the C-terminal region of βII interacts with the N-terminal region of αII, either the wild type, or those with V22F, V22M or V22W mutations. The V22D mutant did not interact with βII. For ITC studies, we used recombinant proteins of the αII N-terminal fragment and of the erythroid beta spectrin (βI) C-terminal fragment; results showed that the Kd values for V22F were similar to those for the wild-type (about 7 nM), whereas the Kd values were about 35 nM for V22M and about 90 nM for V22W. We were not able to detect any binding for V22D with ITC methods. This study clearly demonstrates that the single mutation at position 22 of αII, a region critical to the function of nonerythroid α spectrin, may lead to a reduced level of spectrin tetramers and abnormal spectrin-based membrane skeleton. These abnormalities could cause abnormal neural activities in cells.

Correlation of Different NADPH Oxidase Homologues with Late Endothelial Progenitor Cell Senescence Induced by Angiotensin II: Effect of Telmisartan

Involvement of different NADPH oxidase (NOX) homologues in late endothelial progenitor cell (EPC) senescence induced by angiotensin II (Ang II) remains rarely studied systemically. The goal of our study was to determine NOX homologues which are correlated with late EPCs senescence induced by Ang II. The inhibitory effect of telmisartan was also studied. Late EPCs were obtained from mononuclear cells isolated from peripheral venous blood. Stimulated by Ang II with telmisartan (Tel) or VAS2870 pretreatment or siRNA prior silencing, NOX was detected by RT-PCR and Western blot. Cell senescence was measured by the acidic β-galactosidase activity assay and cell cycle analysis. Intracellular reactive oxygen species (ROS) were analyzed by flow cytometer based on DCFH-DA. A bi-phasic change existed in NOX level after Ang II stimulation. Translocated NOX5 was correlated with early and rapid ROS production, but it contributed little to EPCs senescence. NOX2 and NOX4 were correlated with the late and slow phase and contributed greatly to EPCs senescence. There were no significant changes in NOX1 or NOX3. Telmisartan effectively depressed NOX change and delayed late EPCs senescence. Ang II accelerates late EPCs senescence mainly via increased ROS originating from NOX2 and NOX4 up-regulation or translocated NOX5. Telmisartan effectively inhibited that cascade reaction and delayed EPCs senescence.

Efficacy of TRAIL treatment against HPV16 infected cervical cancer cells undergoing senescence following siRNA knockdown of E6/E7 genes

In this study we investigated E6 and E7 oncogenes from the Human Papilloma Virus as targets for siRNA knockdown in order to boost the efficacy of the anti-cancer drug ‘tumor necrosis factor-related apoptosis inducing ligand’ (TRAIL). SiHa cells were treated with TRAIL following transfection with E6/E7 siRNA and the expression of death receptors DR4 and DR5, cell viability, apoptosis, senescence and cell cycle analysis were undertaken using flow cytometry, MTT viability assay and cellular β-galactosidase activity assays. E6/E7 siRNA resulted in significant upregulation of death receptors DR4 and DR5 but did not result in an enhanced sensitivity to TRAIL. Our results indicate that E6/E7-siRNA induces senescence rather than apoptosis in SiHa cells. The occurrence of senescence in drug resistant cervical cancer cells such as the SiHa cell line by E6/E7 siRNA, among other factors, may prevent TRAIL induced activation of extrinsic and intrinsic pathways that lead to apoptotic cell death. Our findings are significant for combinatorial strategies for cancer therapy since the induction of senescence can preclude apoptosis rendering cells to be recalcitrant to TRAIL treatment.